The invention relates to a thrombin inhibitor comprising an amnoisoquinoline group, a pharmaceutical composition containing the same, as well as the use of said inhibitor for the manufacture of a medicament for treating and preventing thrombin-related diseases.
In literature a large number of peptide-like thrombin inhibitors is disclosed. Most of those thrombin inhibitors contain basic groups at the so-called P1-position, like the basic amino acids arginine and lysine, but also benzamdine and the like. Such a basic moiety is considered essential for antithrombin activity. On the other hand, the basicity of the compounds may impair the uptake of the compounds in the intestines when delivered via the oral route. In WO 98/47876 a class of thrombin inhibitors is disclosed having an aminoisoquinoline moiety as basic group, which show improved trasepithelial transport properties. Within this latter class of compounds a new selection of compounds has now been identified having further improved pharmacological properties.
It has now been found that compounds of the formula (I) 
wherein
R1 is cyclopentyl cyclohexyl or a branched (3-4C)alkyl;
R2 is cyclohexyl or phenyl;
R3 is H or methyl; and
A is an unsubstituted saturated 4, 5 or 6-membered ring;
or a pharmaceutically acceptable salt thereof,
are potent thrombin inhibitors having a significantly increased plasma half-life. Most of the clinical situations in which anti-thrombotic drugs are needed require in general a prolonged half-life (see Sixma, J. J. et al., Thromb. Res. 67; 305-311 (esp. 307), 1992). The compounds of this invention thus are an important improvement in the art.
The compounds of the present invention are useful for treating and preventing thrombin-mediated and thrombin-associated diseases. This includes a number of thrombotic and prothrombotic states in which the coagulation cascade is activated which include, but are not limited to, deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterial occlusion from thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, restenosis following arterial injury or invasive cardiological procedures, postoperative venous thrombosis or embolism, acute or chronic atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention may also be used as anticoagulants in extracorporeal blood circuits, as necessary in dialysis and surgery. The compounds of the invention may also be used as in vitro anticoagulants.
Preferred thrombin inhibitors according to the invention are compounds wherein A is a five-membered ring. Preferably, R2 is cyclohexyl. Other preferred compounds are those, wherein R3 is H. More preferred are compounds wherein R1 is cyclohexyl. The most preferred thrombin inhibitor according to the invention is the compound wherein R1 is cyclohexyl, R2 is cyclohexyl, R3 is H and A is a five-membered ring.
The term branched (3-4C)alkyl means a branched alkyl group having 3 or 4 carbon atoms, e.g. isopropyl.
The invention further includes a process for the preparation of the thrombin inhibitors, including coupling of suitably protected amino acids and aminoisoquinoline derivatives, followed by removing the protective groups.
The compounds according to the formula (I) may be prepared in a manner conventional for such compounds. They may be prepared by a peptide coupling of compounds of formula (II) with compounds of formula (III) using as a coupling reagent for example N,N-dicyclohexylcarbodiimide (DCCI) and 1-hydroxybenzotriazole (HOBT or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), wherein R1, R2, R3 and A have the previously defined meanings. The N-terminus of compounds of formula (II) may optionally carry a protective group such as the t-butyloxycarbonyl group (Boc). The aryl amine group of compounds of formula (III) may optionally carry a protective group such as benzoyl which can be removed after the coupling reaction 
Compounds of formula (II) can be prepared from compounds of formula (IV), wherein Pg1 is a carboxylate protecting group like the benzyl ester, by treatment of compounds of formula (IV) with a appropriate ketone like cyclohexanone or acetone and a reductive agent like sodium triacetoxyborohydride under acidic conditions and thereafter removal of the carboxylate protecting group. 
The compound of formula (III) wherein R3xe2x95x90H (1-amino-6(aminomethyl)isoquinoline) is described in WO 98/47876. The compound of formula (III) wherein R3xe2x95x90Me (1-amino-6-(aminomethyl)-3-methylisoquinoline) can be prepared from 1-amino-6-methoxy-3-methylisoquinoline using the procedures described in WO 98/47876 for the transformation of 1-amino-6-methoxyisoquinoline into 1-amino-7-(aminomethyl)isoquinoline. 1-Amino-6-methoxy-3-methylisoquinoline can be prepared from 3-methoxyphenylacetone using the method described by W. Zielinski and M. Mazik in Heterocycles 38, 375 (1994).
Alternatively, compounds of formula (I) can be prepared from compounds of formula (V), by treatment of compounds of formula (V) with a appropriate ketone like cyclohexanone or acetone and a reductive agent like sodium triacetoxyborohydride under acidic conditions. In this reaction the aryl amine group of compounds of formula (V) may optionally be protected by a group such as benzoyl which can be removed after the reductive amination. 
Compounds of formula (V) can be prepared by a peptide coupling of a dipeptide protected at the N-terminus with a protecting group like the Boc group and compounds of formula (III) using the coupling reagents described before.
Protection of the xcex1-amino functions generally takes place by urethane functions such as the acid-labile tert-butyloxycarbonyl group (Boc), benzyloxycarbonyl (Cbz) group and substituted analogs, the base-labile 9-fluorenylmethyloxycarbonyl (Fmoc) group or the phthaloyl (Phth) group. Other suitable amino protective groups include Nps, Bpoc, Msc, etc. Removal of the protecting groups can take place in different ways, depending on the nature of those protecting groups. Usually deprotection takes place under acidic conditions and in the presence of scavengers. The Cbz group can also be removed by catalytic hydrogenation. An overview of amino protecting groups and methods for their removal is given in The Peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, Eds., (Academic Press, New York, 1981).
Protection of carboxyl groups can take place by ester formation e.g. base-labile esters like methyl- or ethylesters, acid labile esters like tert-butylesters, or hydrogenolytically-labile esters like benzylesters.
The compounds of the invention, which may be in the form of a free base, may be isolated from the reaction e in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts may also be obtained by treating the free base of the formula (I) with an organic or inorganic acid such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, maleic acid, malonic acid, methanesulphonic acid, fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid, and ascorbic acid.
The compounds of this invention possess one or more chiral carbon atoms, and may therefore be obtained as a pure enantiomer, as a pure diastereomer, as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g. crystallization of salts which are obtained from optically active acids and the racemic mixture, or chromatography using chiral columns. For diastereomers straight phase or reversed phase columns may be used.
The compounds of the invention may be administered enterally or parenterally, and for humans preferably in a daily dosage of 0.001-100 mg per kg body weight, preferably 0.01-10 mg per kg body weight. Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the standard reference, Gennaro et al., Remington""s Pharmaceutical Sciences, (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture) the compounds may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection preparation, or as a spray, e.g. for use as a nasal spray.
For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used.
Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.
The elimination half-life and percentage bioavailability of the compounds of the invention may suitably be tested according to the following test in dogs.
The residence time and percentage bioavailability of direct thrombin inhibitors in female Beagle dogs may be measured by determination of the anti-IIa activity in plasma after intravenous or oral administration. In view of the selectivity of the protease inhibitors of the invention, inhibition of thrombin linearly relates with the concentration of the measured protease inhibitor. After intravenous or oral administration of the serine protease inhibitor, blood is collected from the jugular vein at different time-points during the day. After centrifugation of the blood, plasma anti-IIa activities are determined of the plasma samples in a microtiter plate chromogenic assay using a calibration curve of the tested compound itself The obtained data are analysed from the plasma vs time curve e.g. by means of a computerised iterative procedure, based on the Simplex method. Subsequently, the elimination half-lives are calculated using the model of relative error independent of the concentration, and the area under the curve (AUC) is determined with the trapezium rule. Assuming linear kinetics, the percentage bioavailability is calculated by dividing the AUC obtained after p.o. administration by the mean expected normalised AUC after i.v. administration of that dose (X100%).